Structural features of the vitamin K-dependent coagulation proteins will be examined by immunochemical and spectroscopic methods. Conformationally-specific antibodies to native prothrombin and factor X will be isolated by using affinity chromatography using oligopeptides as immunoabsorbants. These peptides will be prepared by solid phase peptide synthesis and by proteolytic cleavage of native proteins. Conformationally-specific antibodies directed against single antigenic determinants on these proteins will be employed using radioimmunassay to detect changes in the conformation of this antigenic site. Specifically, we will evaluate the effect of Ca (II) and/or phospholipid on the conformation of the N-terminal region of these proteins, changes in the conformation of the active site and/or the substrate binding site during zymogen activation, stabilization of tertiary structure by the formation of non-covalent proteins complexes, and the effect of decarboxylation of gamma-carboxyglutamic acid on related and distant regions of the protein. 13C and 31P nuclear magnetic resonance spectroscopy will be employed to establish whether and how many gamma-carboxyglutamic acid residues participate in metal chelation, the structural relationship of phospholipid and metal ions, and the geometry of the metal-binding sites of the vitamin K-dependent proteins. Using gamma-carboxyglutamic acid, derivatives of gamma-carboxyglutamic acid, synthetic peptides containing L-gamma-carboxyglutamic acid, fragments, of prothrombin, and prothrombin, the effect of lanthanide isotropic chemical shift reagents (Pr(III), Dy(III) on atoms in these amino acids and polypeptides as well as bound phospholipid micelles will be examined quantitatively to determine structural features of the Ca(II)-binding site. In sum, these studies will define the structural implications of zymogen conversion in molecular terms, the role of gamma-carboxyglutamic acid in stabilizing protein structure and its contribution to protein function, and the details of the unique class of metal-binding sites present in the vitamin K-dependent coagulation proteins.